How to Make, Run, and Analyze A Gel Electrophoresis

Winnie Lo, Carly Heck, Yuying Ren

Overview ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

DNA is the building block for life. It is important to be able to study DNA to understand and maintain its function. One method that allows DNA to be analyzed is gel electrophoresis; it uses electricity, binding dye, and the polar nature of the genetic material to observe the size of a specific protein fragment. This is possible when DNA is loaded into the porous gel and electricity causes it to migrate. Phosphate groups that make up the genetic material have a negative charge; therefore, they will migrate across the gel from the negative electrode to the positive electrode. The larger the DNA fragment, the slower its migration, so larger pieces will not migrate as far as smaller pieces. Then by using Ethidium Bromide, a DNA­binding dye, the product of gel electrophoresis will fluoresce under UV light and make migration observations possible.

This instruction set has been designed for college students looking to run their own gel electrophoresis. This experiment could fulfill a science project for biology classes or give more experience to students looking to enter a research lab. It requires no previous knowledge of the process, but previous laboratory experience is highly recommended. Overall, this process will take about an hour, 10 minutes to make the gel, 40 minutes to run the gel, and 10 minutes to analyze the gel. After you complete the gel electrophoresis, you will be left with a photograph of your results to analyze further. Be sure to take note of the cautions listed to safely and successfully carry out your experiment.

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Cautions

Ethidium Bromide is a mutagenic dye that is going to be used while running the gel. Please handle this dye with gloves. Direct contact with skin may cause extreme cell proliferation that can lead to cancer or mutated DNA. UV light is needed to analyze the gel at the end of the experiment. Remember to use the UV screen while analyzing the gel to prevent high energy rays from causing eye damage. Do not handle or plug in the wires into the power supplier with wet hands, to avoid electrocution.

Materials ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

The following is the list of supplies will be needed for each task throughout the experiment:

Making the gel:

300 mg Agarose Graduated cylinder Masking tape Microwave 125 mL Erlenmeyer flask Analysis scale Weigh boats Heat protective gloves 1 ul Ethidium Bromide Micropipette Pipette tips Red biohazard bag

Running the gel:

30 mL 1X TAE Buffer 6X loading dye Gel electrophoresis chamber Gel tray Gel caster 160 volts power source Gloves

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Two power­connecting wires Micropipette and tips Wells comb

Analyzing the gel:

Camera UV light source UV screen

Making the Gel ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­ Step 1: Make sure your gel apparatus is properly assembled.

Assembled apparatus unit

Step 2: Weigh 300 mg of agarose and pour into a 125 ml Erlenmeyer flask. Step 3: Measure 30 ml of 1X TAE buffer using a graduated cylinder and pour this into your

flask also. Step 4: Gently swirl to mix the agarose into the buffer.

NOTE: The mixture should look cloudy without any clumps of agarose remaining. Step 5: Place the flask in the microwave for 35 seconds. Step 6: Remove the flask using the heat protective glove and gently swirl again.

NOTE: The gel solution should appear clear with no particles of agarose remaining. If there are particles, microwave for an additional 5 seconds and check again.

Step 7: Let the gel solution cool for approximately 2 minutes.

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Step 8: After cooling, put on a pair of disposable gloves. Using the micropipette, add 1 μl of Ethidium Bromide (orange solution) to the molten agarose and gently swirl. Dispose the pipet tip in a RED biohazard bag. CAUTION: This dye is a mutagen. You must wear disposable gloves when handling the dye and any gel containing this dye.

Step 9: At this point your gel is ready! Bring your solution to the gel apparatus.

Running the Gel ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­ Step 1: Make sure the dams are snug in the apparatus. If the dams are not snug enough, the

molten agarose will leak out before it hardens, which means you have to clean up and start over.

Step 2: Slowly pour the molten agarose into the gel tray.

NOTE: you can remove any big bubbles that might form by using the micropipette to pop them, especially some around the comb.

Step 3: Wait for 10 minutes, and let the agarose harden; once ready, it will appear slightly Blurred and have the appearance of jello.

Agarose gel hardened and ready for samples

Step 4: Carefully remove the comb and dams from the unit, rinse it with water, and return

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them to the storage slots inside the gel box. NOTE: The hardened gel is very fragile; therefore, the comb must be pulled straight up from the gel, in order to preserve the wells without deformation.

Step 5: Pour 1X TAE Buffer into the unit, until it is enough to fill both sides of the apparatus and cover the casted gel.

Step 6: Before you begin to add your samples into the wells, you must mix them with the 6X

loading dye. NOTE: This must be done in order to monitor the sample before UV light can be used.

Step 7: You are now ready to load your samples into the gel! Set a micropipette to 10 ul and

attach a sterile tip. Pipet the sample into each well of the unit. NOTE: This process takes a steady hand, as the wells are very small. Take your time to ensure the sample is contained in the well. It will appear as a small suspended cube in the gel when done correctly.

Step 8: Place the lid firmly on the top of the unit.

NOTE: Make sure the part of the lid with the negative electrode (black) is aligned with the samples.

Step 9: Insert the electrodes into the power supply, matching the red cord with the red plug and

same with black, then turn on the power to 160 volts.

Apparatus with loaded sample and connected power source

Step 10: When the Bromophenol Blue tracking dye has migrated about ½ of the length of your

gel, put on disposable gloves, turn off the power, carefully remove the gel, place it into the tray at your bench, and carry it to the UV light box.

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Analyzing the Gel ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­ Step 1: Place the gel tray on the UV light box in the middle of the blackened glass.

CAUTION: Be sure the UV light box is OFF and is not set to ON until it is covered with the UV screen.

Step 2: Set the UV screen over the UV light source with the sample so that the entire unit is

covered. Step 3: The camera can be placed on the hole in the UV screen to view the gel. Turn on the

camera; open the camera­viewing screen and rotate it until you can see the image. NOTE: 1) be sure the camera is in “Macro” mode. 2) if your gel is dim, you may need to switch to manual mode. NOTE: For the next steps, you need to work quickly to prevent the lens from fogging.

Step 6: Turn on UV light. Make sure hood is completely covering the blackened glass. Step 7: Use camera zoom to fill the frame with the gel, focus the camera, and take a picture.

Photograph of a gel electrophoresis sample

Step 9: Turn off the UV light box. Step 10: After you have photographed your gel, it can be disposed of.

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Troubleshooting ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

While running the gel (Step 10), keep track of the migrating status. There may be a situation where the Bromophenol Blue tracking dye is observed to be stuck in the well and not able to migrate. In this situation, you may turn off the power and check whether the wires are plugged in correctly to the power source. Make sure that the negative electrode (black) is connected on the side of the gel electrophoresis chamber that contains the samples.

Congratulations! You have successfully completed your own gel electrophoresis experiment. ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

Any results or conclusions you wish to draw can be made using the photograph you have taken. In many experiments, scientists will often include this photograph in the actual lab report as supporting evidence.

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Work Cited

DNA Analysis Lab and Review of Lab Skills, BIOL 230W: Biology of Molecules and Cells Laboratory Manual, Fall 2015 Image 1: http://www.biologyreference.com/Dn­Ep/Electrophoresis.html Image of Analysis Scale: www.directindustry.com Image of Pipette tips: www.denvillescientific.com Image of Micropipette: www.indiantradebird.com Image of Gel Electrophoresis Chamber: www.6mgel.com Image of Gel Caster: www.cbsscientific.com Image of UV light: http://www.opsytec.com/fileadmin/_processed_/825a2edc89/ddd3da5d345a8d3fc0d9e36ecb3dc229/uv­table­transilluminator.jpg Image of Agarose Gel: https://upload.wikimedia.org/wikipedia/commons/d/d6/Two_percent_Agarose_Gel_in_Borate_Buffer_cast_in_a_Gel_Tray_(Front,_angled).jpg Image of assembled apparatus: http://www.amtec­lab.com/image.ashx?i=104858.pdf&fn=biorad_Bulletin_2660­Sub­Cell.pdf Image of loaded apparatus: http://sciencefair.math.iit.edu/techniques/gelelectrophoresis/ Image of Wells Combs: http://www.bing.com/images/search?q=gel+electrophoresis+comb&view=detailv2&&id=4A1C1195A901CE273EBCEC74DB82D9585BE2F394&selectedIndex=25&ccid=CVMQqAZH&simid=608015826519917037&thid=OIP.M095310a80647a2d01165cf4bb886980ao0&ajaxhist=0

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